Project Summary/Abstract Drug addiction is a complex brain disorder that takes an enormous toll on individual and economic health. Current drug-abuse treatments are not effective in all individuals, and many recovering addicts continue to relapse. Drug exposure usurps normal reward circuit function, including the connections between the ventral tegmental area (VTA) and the nucleus accumbens (NAc). Both NAc and VTA undergo molecular and physiological changes in response to drugs of abuse. The VTA sends dopaminergic projections to the NAc, and the NAc sends GABAergic projections back to the VTA. Activity in NAc and VTA are key for driving drug use and are heavily implicated in drug relapse. Numerous studies have established the importance of the dopaminergic VTA to NAc projection, however the connection from NAc back to VTA is understudied in drug addiction. The incidence of opioid use, opioid addiction, and death from opioid overdose are at an unprecedented high. In this K99/R00 Pathway to Independence Award, I aim to identify how NAc projections to the VTA influence opioid use and relapse. Using a mouse model of fentanyl self-administration, I have collected preliminary data showing NAc neurons that project to VTA control fentanyl seeking after forced abstinence. In this study, I intend to utilize intersectional circuit manipulation and cutting-edge molecular biology to investigate the molecular mechanisms by which NAc projections to VTA control fentanyl intake and relapse. During the mentored phase, in Aim 1, I will learn operant self-administration procedures and combine them with chemogenetic manipulation to assess the necessity of NAc terminals in VTA in fentanyl use and relapse. In Aim 2, I will learn RNAseq and in situ hybridization to profile molecular adaptations in specific VTA neurons after fentanyl self-administration. During the independent phase, in Aim 3, I will use novel viral constructs to assess the role of molecular manipulations in specific VTA neurons in fentanyl use and relapse. In Aim 4, I will assess the role of fentanyl and molecular manipulations on dopamine release in the NAc using fast-scan cyclic voltammetry. Together, the research proposed in this Pathway to Independence Award will elucidate molecular mechanisms in the NAc-VTA circuit driving opioid use and relapse, while simultaneously providing me with the tools necessary for establishing an independent research program that bridges molecular biology, circuit manipulation, operant behavior, and voltammetry for examining addiction.